Electrochemical cell

ABSTRACT

An electrochemical cell for detection and quantification of analytes in a liquid sample, particularly a liquid sample having a small volume. In a preferred embodiment, the electrochemical cell comprises an assembly of conducting layers and insulating layers. The electrochemical cell can be formed by depositing conducting materials and insulating materials in alternating layers on an insulating substrate. It is preferred that the layer furthest from the insulating substrate be an insulating layer to minimize the damage of the conducting layers during handling of the electrochemical cell. In another embodiment, the assembly of conducting layers and insulating layers can be formed on both major surfaces of the insulating substrate. The assembly can comprise at least one working electrode and at least one other electrode, e.g., a dual-purpose reference/counter electrode.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to electrochemical cells. More specifically, theinvention relates to electrochemical cells suitable for the detectionand measurement of concentration of analytes in liquid samples.

2. Discussion of the Art

For conventional electrochemical analysis of a liquid sample, electrodesare dipped in the sample for electrochemical determination of the typeof analyte or measurement of the concentration of analyte or both. Theelectrodes are spaced apart from each other, and the electrolytes in thesample provide ionic communication between the electrodes. In a majorityof situations, the sample is static during measurement; in someinstances, the sample flows through an electrochemical detector when thesample is in a fluid motion, such as in the case of flow injectionanalysis. The dimensions of the electrodes define the volume of thesample required for the measurement. The constraints relating to thevolume of the sample and the requirement of rapid measurement may callfor the use of microelectrodes, when the volume of the sample is notsufficient to cover the surface area of electrodes of conventional size.

Different methods of forming microelectrodes for the fabrication ofelectrochemical cells have been demonstrated. Interdigitated electrodesor band electrodes can be formed, with the electrodes being in closeproximity to minimize the volume of sample required to perform anelectrochemical measurement. In these devices, the electrodes arepositioned on the same surface. U.S. Pat. No. 5,045,828 describes ahumidity sensor comprising (a) a substrate having an electricallyinsulating surface; (b) a pair of spaced electrodes on the surface; and(c) a film having a thickness of approximately 5 microns or less on thesurface interconnecting the electrodes. Conventional biosensors have aworking electrode and a dual-purpose reference/counter electrode on thesame major surface of an insulating substrate. The reactive chemistry ispositioned on either the working electrode or on both the dual-purposereference/counter electrode and the working electrode. U.S. Pat. No.5,509,410 describes a sensor system adapted for releasable attachment tosignal readout circuitry. The strip comprises an elongated supportadapted for releasable attachment to readout circuitry; a firstconductor and a second conductor each extending along the support andcomprising means for connection to the circuitry. An active electrode,positioned to contact a liquid mixture and the first conductor,comprises a deposit of an enzyme capable of catalyzing a reactioninvolving the compound and preferably an electron mediator, capable oftransferring electrons between the enzyme-catalyzed reaction and thefirst conductor. A reference electrode is positioned to contact themixture and the second conductor. The system includes circuitry adaptedto provide an electrical signal representative of the current.

WO 03/05639 discloses a microelectrode in the form of a receptacle. Thereceptacle comprises a working electrode in the wall of the receptacle,typically having a small surface area. A counter electrode is alsopresent, the electrode typically having a much larger surface area thanthat of the working electrode, generally a surface area which is atleast an order of magnitude larger than that of the working electrode.The electro-active substance may be placed into the receptacle and isoptionally dried into position. The sample is then applied to thereceptacle in order that testing can be carried out. The electro-activesubstance will typically not contact the working electrode in the wallof the receptacle during storage and therefore fouling of this electrodeis minimized.

Various references in the prior art describe methods of fabrication ofelectrochemical cells for various analytical applications. Some of thesereferences describe electrochemical cells having electrodes positionedside-by-side and having reagents on the surfaces of the electrodes,while other of the references describe electrochemical cells having areceptacle having one of the electrodes along the wall of anelectrochemical cell and reagents positioned away from the activeelectrode. The positions and dimensions of the electrodes constitutingthe cell determine the volume of the electrochemical cell. Therefore, itwould be desirable to provide electrochemical cells where the electrodesare positioned in such a manner as to decrease the volume of liquidsample required by the cell, in which positioning of reagents can be incontact with the working electrode.

SUMMARY OF THE INVENTION

This invention provides an electrochemical cell for detection andquantification of analytes in a liquid sample, particularly a liquidsample having a small volume.

In a preferred embodiment, the electrochemical cell comprises anassembly of conducting layers and insulating layers. The electrochemicalcell can be formed by depositing conducting materials and insulatingmaterials in alternating layers on an insulating substrate. It ispreferred that the layer furthest from the insulating substrate be aninsulating layer to minimize the damage of the conducting layers duringhandling of the electrochemical cell. A passage can be formed throughthe conducting layers and the insulating layers, either including or notincluding the insulating substrate, to expose the edges of the layers,which collectively form the wall or walls of the passage. The exposededges of the conducting layers form the electrodes of theelectrochemical cell. The electrochemical cell comprises at least oneworking electrode and at least one other electrode, e.g., a dual-purposereference/counter electrode. Alternatively, the electrochemical cell cancomprise at least one working electrode, one reference electrode, andone counter electrode. The shape and the dimensions of the passage canbe selected to optimize the area of the exposed electrodes and thevolume of the electrochemical cell. As used herein, the term “optimize”refers to the process of maximizing the surface area of the electrodes,while minimizing the volume of the liquid sample, so as to obtain anaccurate electrical response with a very small liquid sample.

In another embodiment, the assembly of conducting layers and insulatinglayers can be formed on both major surfaces of the insulating substrate.The assembly can comprise at least one working electrode and at leastone other electrode, e.g., a dual-purpose reference/counter electrode.Alternatively, the electrochemical cell can comprise at least oneworking electrode, one reference electrode, and one counter electrode.It is preferred that the electrochemical cell have insulating layersoverlying the major surfaces of the conducting layers not facing theinsulating substrate to minimize the damage of the conducting layersduring handling of the electrochemical cell.

The number of conducting layers in the assembly determines the number ofelectrodes in the electrochemical cell. The conducting layersfunctioning as working electrodes preferably contain reagent(s) specificto one or more analytes in the liquid sample or support areagent-containing layer containing reagent(s) specific to one or moreanalytes in the liquid sample, such as, for example, glucose, ketonebodies, lactate etc. One or more of these conducting layers can also beused to determine the interference from electroactive species that maybe present in the sample. At least one of these conducting layers mustcarry out the function of a reference electrode. Optionally, theelectrochemical cell can contain a counter electrode, separate anddistinct from a reference electrode.

The volume of liquid sample(s) that can be introduced into theelectrochemical cell is determined by the cumulative thickness of theindividual layers and the perimeter of the passage(s). More than onepassage can be formed in the electrochemical cell to provide a pluralityof electrochemical cells in an assembly of conducting layers andinsulating layers. In these situations, all the passages can be used toperform a plurality of identical assays for the same set of analyteswith a single liquid sample to increase the sensitivity of the assay, orall the passages can be used to perform a plurality of identical assaysfor the same set of analytes, but with different liquid samples. Aplurality of passages can also be used for the analyses of differentanalytes with a single liquid sample. The locations of the passages canoperate to either minimize the volume of sample or to minimize crosstalk, depending on the application.

The invention also provides a method for constructing electrochemicalcells that can operate with small volumes of sample. The electrochemicalcell of this invention can be constructed by interlaying conductinglayers and insulating layers and then forming a passage to expose theedges of the layers to a liquid sample. The conducting layers exposed tothe liquid sample form the electrodes of an electrochemical cell. In thepassage, adjacent conducting layers, i.e., electrodes, are separated byan insulating layer. Specificity to the electrochemical cell can beprovided by incorporating a reagent that specifically reacts with ananalyte of interest, thereby generating a measurable signal.

The reagents specific for an analyte can be applied at the same time asthe layer of conducting material in the form of a discrete layer,wherein the appropriate reagent(s) is (are) present in a layer ofconductive material forming an electrode; alternatively, the reagent(s)can be applied as a layer impregnated with reagent(s), the applied layerbeing separate from the layer forming the electrode; as a furtheralternative, the reagent(s) can be coated along the wall or walls of apassage.

The electrochemical cells of this invention can be used for any type ofelectrochemical measurement. The conducting layers can be modified tomeasure a specific analyte. The electrochemical cell can utilize ionsensitive electrodes. In addition, the electrochemical cell can be anelectrochemical biosensor having the appropriate reagent(s) in aconducting layer that is specific to an analyte of interest.

This invention makes it possible to prepare electrochemical cells thatrequire extremely low volumes of sample. The electrochemical cells ofthis invention can be reproduced with great accuracy and precision.Assays for a single analyte or a plurality of analytes can be performedwith the electrochemical cell of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of one embodiment of theelectrochemical cell of this invention.

FIG. 2 is a side view in elevation of a section taken along line 2-2 ofthe electrochemical cell of FIG. 1.

FIG. 3 is an end view in elevation of a section taken along line 3-3 ofthe electrochemical cell of FIG. 2.

FIG. 4 is a top plan view of an electrochemical cell of FIG. 1.

FIG. 5 is an exploded perspective view of one embodiment of theelectrochemical cell of this invention.

FIG. 6 is a side view in elevation of a section taken along line 6-6 ofthe electrochemical cell of FIG. 5.

FIG. 7 is an end view in elevation of a section taken along line 7-7 ofthe electrochemical cell of FIG. 6.

FIG. 8 is a top plan view of an electrochemical cell of FIG. 5.

DETAILED DESCRIPTION

As used herein, the expression “electrochemical cell” refers to a devicecomprising a working electrode and a counter electrode which areconnected to one another electrically. When in use, electrochemicalreactions occurring at each of the electrodes cause electrons to flow toand from the electrodes, thus generating a current. An electrochemicalcell can be set up either to harness the electrical current produced,for example in the form of a battery, or to detect electrochemicalreactions which are induced by an applied current or voltage.

As used herein, the term “layer” means a single thickness, coating, orstratum that covers a surface. The expression “major surface” means thesurface of a substrate that has a larger area than another surface. Aplanar substrate will have two major surfaces and at least one minorsurface. The term “passage” means a path, channel, or duct through whicha liquid can pass. In the invention described herein, a passage can runthrough all the layers, including the substrate, or can run through lessthan all of the layers. The term “volume” means the volume of a liquidrequired to fill a single passage or a plurality of passages. Anelectrochemical cell can have a single passage or a plurality ofpassages. The term “aperture” means an opening into which a liquidsample can enter the passage or a segment of the passage. The passagehas a depth and the aperture has an area.

The expression “working electrode” means an electrode where the reactionof interest takes place. The current is proportional to theconcentration of an analyte, e.g., glucose, at the working electrode;the expression “reference electrode” refers to an electrode thatmeasures the potential at the interface of the working electrode and thesample as accurately as possible; the expression “counter electrode”refers to an electrode that ensures that the correct potentialdifference between the reference electrode and the working electrode isbeing applied; a “dual-purpose reference/counter electrode” is anelectrode that acts as a reference electrode as well as a counterelectrode. In an ideal situation, no current passes through thereference electrode. The potential difference between the workingelectrode and the reference electrode is assumed to be the same as thedesired potential at the working electrode. If the potential measured atthe working electrode is not the potential desired at the workingelectrode, the potential that is applied between the counter electrodeand the working electrode is altered accordingly, i.e., the potential iseither increased or decreased. The reaction at the counter electrode isalso equal and opposite to the charge transfer reaction occurring at theworking electrode, i.e., if an oxidation reaction is occurring at theworking electrode then a reduction reaction will take place at thecounter electrode, thereby allowing the sample to remain electricallyneutral. No current passes through an ideal reference electrode, andsuch an electrode maintains a steady potential; current does passthrough a dual-purpose reference/counter electrode, and thus, thedual-purpose reference/counter electrode does not maintain a steadypotential during the measurement. At low currents and/or at shortdurations of time for measurement, the shift in potential is smallenough such that the response at the working electrode is notsignificantly affected, and hence the dual-purpose reference/counterelectrode is designated a dual-purpose reference/counter electrode.

The expression “conducting layer” means the electrically conductinglayer that is interposed between two insulating layers. The expression“insulating layer” means either a layer that is interposed between twoconducting layers or a layer that overlies a conducting layer, one majorsurface of the insulating layer being in contact with one major surfaceof the conducting layer. The resistance of the insulating layer issufficiently high that current does not flow through the insulatinglayer.

The term “reagent(s)” means substance(s) that is (are) an activecomponent(s) of the detection and quantification process, whereby thepresence or concentration of an analyte in a sample is determined.Reagents include, but are not limited to, enzymes, mediators,co-enzymes, ionophores, cells, or combinations of the foregoing. Thereagents typically comprise an enzyme and a mediator. A mediator is achemical species that has two or more oxidation states of distinctelectro-active potentials that allow a reversible mechanism oftransferring electrons/charge to an electrode. The enzyme reacts withthe analyte in the sample, thereby catalyzing oxidation of the analyte.The enzyme is reduced in the oxidation reaction, and the reduced enzymeis regenerated by the mediator. Representative examples of enzymesinclude glucose oxidase, lactate oxidase, beta hydroxybutyratedehydrogenase, and the like. Representative examples of mediatorsinclude ferrocene, ferricyanide, quinones, and the like. Alternatively,ionic species and metal ions can be used in place of the enzyme to formelectrochemically detectable compounds when they react with the analyte,such as ionophores used for the ion-sensitive electrodes.

Referring now to FIGS. 1-4, an electrochemical cell 10 comprises aninsulating substrate 12, a plurality of conducting layers 14 a, 14 b,and an insulating layer 16 a interposed between the two conductinglayers 14 a and 14 b. Another insulating layer 16 b overlies theconducting layer furthest from the insulating substrate 12. Conductivetracks 18 a, 18 b are applied to the insulating substrate 12, andconductive tracks 18 c, 18 d are applied over the conductive tracks 18a, 18 b, respectively. A layer of reagent(s) 20 overlies the conductinglayer 14 a. A passage 22 passes through (a) the insulating substrate 12,(b) the plurality of conducting layers 14 a, 14 b, (c) the plurality ofinsulating layers 16 a, 16 b, and (d) the layer of reagent(s) 20. Theedges 24, 26 of the conducting layers 14 a, 14 b, respectively, the edge28 of the layer of reagent(s) 20, and the edges 30, 32 of the insulatinglayers 16 a, 16 b, respectively, form the wall of the passage 22. Theconducting layers 14 a, 14 b form the electrodes of the electrochemicalcell 10. In the embodiment shown in FIGS. 1-4, there are two insulatinglayers 16 a, 16 b, and two conducting layers 14 a, 14 b in addition tothe insulating substrate 12.

Referring now to FIGS. 5-8, an electrochemical cell 10′ comprises aninsulating substrate 12′, a plurality of conducting layers 14 a′, 14 b′,and a plurality of insulating layers 16 a′, 16 b′, the insulatingsubstrate 12′ interposed between the two conducting layers 14 a′, 14 b′.The insulating layers 16 a′, 16 b′ overlie the conducting layers 14 a′,14 b′, respectively. Conductive track 18 b′ is applied to one majorsurface of the insulating substrate 12′, and conductive track 18 c′ toanother major surface of the insulating substrate 12′. A passage 22′passes through the insulating substrate 12′, the plurality of conductinglayers 14 a′, 14 b′, and the plurality of insulating layers 16 a′, 16b′. The edges 24′, 26′ of the conducting layers 14 a′, 14 b′,respectively, and the edges 28′, 30′ of the insulating layers 16 a′, 16b′, respectively, form the wall of the passage 22′. The conductinglayers 14 a′, 14 b′ form the electrodes of the electrochemical cell 10′.In the embodiment shown in FIGS. 5-8, there are two insulating layers 16a′, 16 b′ and two conducting layers 14 a′, 14 b′ in addition to theinsulating substrate 12′. It is preferred that the conducting layer 14a′ contain at least one reagent suitable for an assay for determiningthe presence or concentration of an analyte of interest. Areagent-containing layer (not shown) separate from the conducting layer14 a′ can be placed so as to be in face-to-face contact with theconducting layer 14 a′ to supply any reagent(s) not present in theconducting layer 14 a′, if the conducting layer 14 a′ does not containall of the reagent(s) needed to carry out the assay.

Materials that are suitable for the insulating substrate include, butare not limited to, polymeric materials, such as, for example, polyvinylchloride, polycarbonate, polyester, and the like. These materials arecommercially available. The purpose of the insulating substrate is toprovide mechanical support for the layers overlying the substrate.

Materials that are suitable for the conducting layers are electricallyconductive and include, but are not limited to, carbon, metals, such as,for example, gold, palladium, platinum, copper, silver, electricallyconductive compounds, such as, for example, silver chloride, andsemi-conducting materials, such as, for example, indium doped tin oxide.In some instances, more than one conductive material can be mixed toform a conducting layer; in other instances, a conducting layer can beprepared by overlying one conducting material with another conductingmaterial.

The conducting layer can be formed by depositing an electricallyconductive material on an insulating layer by means of conventionaltechniques, such as, for example, screen printing, vapor deposition, inkjet printing, etc.

The electrochemical cell can also contain at least one additionalconducting layer and an insulating layer for each additional conductinglayer. One of the additional conducting layers can form a counterelectrode. As stated previously, additional conducting layers canfunction as working electrodes. These additional conducting layersfunctioning as working electrodes allow different measurements to becarried out on the same sample by applying different potentials acrosstwo or more of the conducting layers functioning as workingelectrode/counter electrode pairs. Alternatively, the same potential maybe applied to each conducting layer functioning as a working electrodeand the same measurement recorded several times for the same sample.This procedure helps to eliminate or detect errors in the measurementstaken.

Additional working electrodes can be employed in the electrochemicalcell for one or more of the following functions:

-   -   1) As a second working electrode to determine the same analyte        as the first working electrode by increasing the surface area of        the working electrode;    -   2) As a second working electrode to determine the same analyte        as the first working electrode, whereby the integrity of the        measurement (as a counter check on the first measurement) is        verified;    -   3) As a second biosensor for an analyte to measure a second        analyte, different from the first analyte, in the liquid sample;    -   4) As a means for measuring the background signal to compensate        for the interfering agents in the liquid sample.        The additional working electrode can use the same dual-purpose        reference/counter electrode as the first electrode or can have        its own dual-purpose reference/counter electrode.

The insulating layer provides a separation between two conducting layersto prevent short circuits. The material for the insulating layer istypically a polymer, such as, for example, an acrylate, polyurethane,polyolefin, polyester, e.g., polyethylene terephthalate, or the like.Polycarbonate and other plastics and ceramics are also suitable asmaterials for the insulating layer. The insulating layer can be formedby evaporating a solvent from a solution of the polymer. Liquids thatharden after application can also be used, e.g., varnishes.Alternatively, cross-linkable polymer solutions can be used. These canbe cross-linked by exposure to heat or electromagnetic radiation or bymixing together the active parts of a two-component cross-linkablesystem. Dielectric inks can also be used to form insulating layers. Apreferred material for the insulating layers is commercially availableunder the trademark “POLYPLAST” (Sericol Ltd., Broadstairs, Kent, UK).The insulating layer can be deposited over a given area of theconducting layer in such a manner to leave a portion of the conductinglayer exposed in order to provide electrical contacts so that theelectrochemical cell can be connected to an apparatus for measuring theelectrochemical response, such as voltage difference (in mV) or current(in amperes). The insulating layer can be deposited by any method in theart, such as, for example, screen-printing, laminating, or otherconventional chemical depositing techniques. A preferred insulatinglayer can be formed by using a preformed polymeric suspension, such asone designated by the trademark Sericard® (Sericol Ltd., Broadstairs,Kent, UK), or a monomeric solution that is polymerized after beingapplied.

At least two conducting layers are required, one in order to function asthe working electrode and another in order to function as the referenceelectrode of the electrochemical cell. If only two conducting layers areused, one conducting layer can function as the working electrode and theother conducting layer can function as a dual-purpose reference/counterelectrode. The electrochemical cell can contain a third conductinglayer, which will function as the counter electrode. A plurality ofworking electrodes can be defined by utilizing additional conductinglayers. These working electrodes can be used for the measurement of thepresence or the amount or both the presence and the amount of a singleanalyte or of a plurality of analytes in a given sample.

A conducting layer that functions as a working electrode is preferablyformed from carbon, palladium, gold, or platinum, for example, in theform of conductive ink. The conductive ink may contain additionalmaterials, such as, for example, platinum, or graphite, or both platinumand graphite. Two or more layers may be used to form a workingelectrode, the layers being formed of the same or different materials.

A conducting layer that functions as the dual-purpose reference/counterelectrode, reference electrode, or counter electrode is preferablyformed from carbon, palladium, gold, or platinum, Ag/AgCl, for example,in the form of conductive ink. The conductive ink may contain additionalmaterials, such as, for example, platinum or graphite or both. Two ormore layers may be used to form the dual-purpose reference/counterelectrode, the layers being formed of the same or different materials.In the case where three conducting layers are employed, one of theconducting layers can function as a working electrode, one of theconducting layers can function as a reference electrode, and one of theconducting layers can function as a counter electrode.

The number of conducting layers in the electrochemical cell determinesthe number of electrodes in the electrochemical cell. These conductinglayers preferably contain reagent(s) specific to one or more analytes inthe sample, such as, for example, glucose, ketone bodies, lactate etc.,or are adjacent to a layer containing reagent(s) specific to one or moreanalytes in the sample, such as, for example, glucose, ketone bodies,lactate etc. One or more of these conducting layers can also be used todetermine interference from electroactive species that may be present inthe sample. At least one of these conducting layers must function asreference electrode. Optionally, the electrochemical cell can alsocontain a conducting layer that functions as a counter electrode.

In the method of this invention, the liquid sample can be a sample ofwhole blood. In other electrochemical cells suggesting the use of threeelectrodes, the liquid sample can be whole blood that has been filteredor treated to remove red blood cells or other hemocytes.

There are numerous ways to prepare the electrochemical cell of thisinvention. In one embodiment, an insulating support is coated with aconducting material, such as carbon or conductive metal, by means ofscreen-printing or other deposition technique, such as sputtering, toform a first conducting layer. The reagent(s) is (are) then applied overthe conducting layer by any method of application, such as, for example,drop coating, screen-printing, ink jet printing, or chemical attachmentof the reagent to the layer of conducting material, such as, forexample, by means of a chemical linking group. One can also apply thereagent(s) by in situ polymerization of monomers, such as, for example,pyrrole or acrylamide, in the presence of reactive components, such as,for example, enzyme or mediator, thereby resulting in the physicalentrapment of the reactive component in a polymeric matrix. Thereagent(s) is (are) located in the region of the electrochemical cellthrough which the passage is to be formed. The area of the reagent layeris greater than the area of the aperture of the passage. An insulatinglayer is then deposited over the first conducting layer and reagentlayer in such a manner that a part of the first conducting layer isexposed to enable removable contact with a measurement device. A secondconducting layer is then applied over the insulating layer in such amanner as to leave the contact area of the second conducting layer,i.e., the dual-purpose reference/counter electrode, exposed. Aninsulating layer is applied over the second conducting layer in such amanner as to leave the contacts exposed. A passage is then formed bycutting through the insulating layers and the conducting layers and, ifdesired, through the insulating substrate.

The passage can be formed by cutting though the layers by any method,including but not limiting to, punching, die-cutting, milling, drilling,ablating, laser cutting, etc. One of ordinary skill in the art canreadily choose an appropriate method, based on the physical propertiesof the layers and the expected use of the electrochemical cell. Withrespect to punching, a single passage can be punched from either the topof the electrochemical cell assembly or from the bottom of theelectrochemical cell assembly. Overlapping double passages can bepunched from either the top of the electrochemical cell assembly or fromthe bottom of the electrochemical cell assembly. Laser-cutting by meansof ultraviolet radiation provides a higher yield than do other methods.Care should be taken to ensure that the mechanical pressure experiencedby the layers during the formation of the passage does not result inelectrical conductivity between two adjacent conducting layers. It isalso envisioned that the physical step of forming the passage can befollowed by a chemical etching process to create patterns that wouldenhance the surface area of the electrodes.

In an alternative embodiment, a layer containing a mixture of silver andsilver chloride is printed on one major surface of an insulatingsubstrate. The insulating substrate is preferably polyvinyl chloride(PVC), Melinex® polyester (E. I. duPont de Nemours, Inc.). This layer iscovered with an insulating layer, but leaving a contact area forremovable connection with the measurement device. The insulating layeris preferably made of Sericard® material. The other major surface of theinsulating substrate is then coated with a first conducting layer. Thefirst conducting layer preferably comprises carbon or a conductivemetal. Coating is carried out by screen-printing or another technique,such as sputtering. The reagent(s) is (are) then printed over the firstconducting layer by any deposition method, such as, for example, dropcoating, screen printing, ink jet printing, etc., in the area where thepassage is to be formed, such that dimension of the area where thereagent(s) is (are) deposited is greater than the dimension of theaperture of the passage. An insulating layer is then applied to coverthe reagent layer and the first conducting layer, while allowing acontact area to be exposed for removable connection with the measurementdevice. A passage is then formed in such a manner that the passagepasses through the insulating layers, insulating substrate, and theconducting layers, as well as the reagent.

The electrochemical cell of this invention can be prepared by aphotopolymerization technique in which a photopolymerizable material isapplied over a conducting layer and the portion of thephotopolymerizable material to be retained in the final article is curedby means of the appropriate application of electromagnetic radiation.After the desired number of conducting layers and photopolymerizablelayers are applied, the uncured portions of the photopolymerizablelayers are removed by washing in an appropriate solvent.

The dimensional parameters of the layers are affected by the method ofapplying the layer to a substrate or to an adjacent layer. For example,screen-printing typically provides a thickness of from about 2 μm toabout 100 μm, depending on the screen mesh and the physical propertiesof the material being applied. Sputtering typically provides a thicknessof from about 10 nm to about 10 μm. Lamination can provide a thicknessof from about 25 μm to about 6 mm. The insulating layers and theconducting layers must be made of material having sufficient rigidity toavoid being excessively compressed under mechanical forces, whichexcessive compression would result in variations in the thickness of theelectrodes as well as increasing the possibility of bringing about shortcircuits.

Materials that can be used as reagents, either in a reagent-containinglayer or incorporated into the material of a conducting layerfunctioning as a working electrode, include enzymes, such as glucoseoxidase, glucose dehydrogenase, beta-hydroxybutyrate dehydrogenase,lactate dehydrogenase, etc., and a coenzyme, such as, for example,nicotinamide adenine dinucleotide (NAD), if required. Thereagent-containing layer can further include an oxidation-reductionmediator.

The reagent(s) of the electrochemical cell need not be introduced to theelectrochemical cell by way of a reagent layer. The reagent(s) can beapplied along the wall of the passage after the passage is created. Thereagent(s) can be provided in a porous material wherein the porousmaterial is positioned so as to fill the cavity surrounded by the wallof the passage.

If used, the amount(s) of reagent(s) required in the electrochemicalcell are not critical, and the precise amount(s) of reagent(s) to beused for desired performance can readily be determined by one ofordinary skill in the art.

A passage for receiving the liquid sample can be formed through thevarious layers of the electrochemical cell. The exposed edges of theconducting layers forming a portion of the wall of the passage definethe electrodes of the electrochemical cell. The cross-section of thepassage can have any shape, e.g., circular, elliptical, polygonal. Theshapes can be regular, e.g., equilateral triangle, square, or irregular,e.g., polygon having sides of differing lengths. In addition, the shapeof the cross-section of the passage can vary along the length of thepassage. Furthermore, each layer can have an aperture of a differentshape. It is preferred that the surface areas of the electrodes exposedto the passage be optimized to obtain the desired signal to noise ratio.In general, the higher the surface area, the better the signal to noiseratio. The passage preferably includes at least one opening to serve asa vent to enable the passage to be filled with liquid easily. Theopening is preferably formed in the insulating substrate. If such anopening(s) is (are) not present, the sample may not enter the passagewhen it flows into the aperture, or it may enter the passage only withdifficulty. The opening(s) can be smaller than the aperture, but shouldbe large enough to allow air to escape from the electrochemical cell.

In some embodiments, a passage need not be formed. In this type ofembodiment, an end of the electrochemical cell is placed in contact withthe liquid sample. The end of the electrochemical cell that contacts theliquid sample is characterized by having the edges of the conductinglayers exposed.

In assays where an electroactive species in a liquid sample is measuredwithout the need for any reagent at all, the conducting layerconstituting the working electrode need not have any reagent depositedthereon. As is well-known, electrochemical measurement is carried out byusing a working electrode coupled to a reference electrode. Themeasurement can involve a change in the potential (potentiometry) or thegeneration of current (amperometry). The electrodes by themselves do notexhibit specificity to an analyte. The specificity can be imparted tothe electrode by having an enzyme (in the case of biosensor) that reactswith only one of a plurality of analytes in a mixture of analytes or byemploying a filtration technique that would selectively allow only oneof a plurality of analytes in a mixture to pass through a filtrationdevice. In electrochemical measurements of certain analytes, such asdopamine in the brain, the determination of interfering agents in a“dummy” electrode of a biosensor is one example wherein anelectrochemical measurement is carried out without the use of anyreagent on the surface of the working electrode. See, for example, U.S.Pat. No. 5,628,890.

OPERATION

Any method of introducing liquid samples to the electrochemical cell canbe used. The dimensions of the passage suitable for uptake of sample bycapillary attraction can be specified. Other methods, such as, forexample, gravitational forces, chemically-aided wicking, or suction bymeans of vacuum, can be used. In certain applications, the passage canbe filled with a porous material that will allow uptake of the sample bywicking.

The aperture(s) in the passage can be designed to allow theelectrochemical cell to be integrated with a device for extractingliquid biological samples from a subject. For example, a mechanicaldevice, such as a lancet, or an optical device, such as a laser, can bedirected at the sample extraction site through the aperture(s) of thepassage to create an artificial opening in a human body (skin). Theliquid sample emerging from the artificial opening can then betransferred to the electrochemical cell either by an additionalmechanical force, such as, for example, suction provided by vacuum, orby a naturally provided force, such as, for example, gravitation. Adevice that is suitable for integrating the electrochemical cell of thisinvention is described in U.S. Pat. No. 6,093,156, incorporated hereinby reference.

The electrochemical cell can be used as a flow cell, with liquidtraversing the length of the passage under convection, diffusion, orosmosis. Based on the dimensions of the aperture(s) of the passage,larger species from the sample can be excluded. Representative examplesof larger species include, but are not limited to, cells, protein, andskin.

The volume of a given passage specifies the volume of liquid samplerequired by that passage of the electrochemical cell. The overall volumeof a given passage is equal to the sum of the volumes of each section ofthat passage. The volume of liquid required to fill a given passage inthe electrochemical cell is determined by the cumulative thickness ofthe individual layers and areas of the various sections of that passage.It is preferred that any given passage not have a volume exceeding 1microliter.

More than one passage can be formed in an electrochemical cell to form,in effect, a plurality of electrochemical cells in the assembly. Inthese situations, a plurality of passages can be used in a plurality ofidentical assays with one liquid sample to increase the sensitivity ofthe assay; alternatively, a plurality of passages can be used to performa plurality of identical assays for the same analyte with differentliquid samples or to perform assays for a plurality of analytes with asingle liquid sample. The locations of the passages can be specified toeither minimize the volume of sample required or to minimize cross talk,depending on the application. As the passages are moved farther apart,crosstalk is reduced. As the passages are moved closer together, a lowervolume of sample is required.

Measuring devices that are suitable for use in this invention includeany commercially available analyte monitor that can accommodate anelectrochemical cell having a working electrode and a dual-purposereference/counter electrode. Alternatively, an analyte monitor that canaccommodate an electrochemical cell having a working electrode, areference electrode, and a counter electrode can be used. Such analytemonitors can be used to monitor analytes, such as, for example, glucoseand ketone bodies. In general, such a monitor must have a power sourcein electrical connection with the working electrode, the referenceelectrode, and the counter electrode. The monitor must be capable ofsupplying an electrical potential difference between the workingelectrode and the reference electrode of a magnitude sufficient to causethe electrochemical oxidation of the reduced mediator. The monitor mustbe capable of supplying an electrical potential difference between thereference electrode and the counter electrode of a magnitude sufficientto facilitate the flow of electrons from the working electrode to thecounter electrode. In addition, the monitor must be capable of measuringthe current produced by the oxidation of the reduced mediator at theworking electrode.

In a measurement employing the electrochemical cell of this invention, aconstant voltage is applied at the working electrode and the current ismeasured as a function of time. This technique is known aschronoamperometry. The voltage applied should be equal or higher to thevoltage required to oxidize the reduced mediator. Thus, the minimumvoltage required therefore is a function of the mediator.

The sample is responsible for the solution resistance. The solutionresistance inhibits the flow of electrons. The effect of solutionresistance on the measurement is minimized by this invention. Arrangingthe electrodes close together obviously minimizes the effect of solutionresistance because solution resistance is a function of the spacingbetween the electrodes. By allowing the current to flow through adifferent electrode, the effect of solution resistance on the workingelectrode can be minimized.

In an amperometric measurement, the current should decay with timeaccording to the Cottrell equation.

$i_{t} = \frac{{nFAC}_{o}D_{o}^{1/2}}{\pi^{1/2}t^{1/2}}$where

i_(t)=the current at time t

n=number of electrons

F=Faraday's constant

A=area of the electrode

C_(o)=bulk concentration of the electrochemically active species

D_(o)=diffusion coefficient of the electrochemically active species.

Therefore, i_(t)t^(1/2) should be a constant.

In an amperometric measurement, a constant voltage is applied at theworking electrode with respect to the reference electrode, and thecurrent between the working and counter electrodes is measured. Theresponse of the electrochemical cell has two components, catalytic(glucose response component) and Faradaic (solution resistancecomponent). If the resistance of the solution is minimized, the responseof the electrochemical cell at any given time will have substantiallyhigher glucose response component, as compared with the solutionresistance component. Therefore, one is able to obtain good correlationwith the concentration of glucose from the response of theelectrochemical cell even at assay times as short as one second. If theresistance of the solution is high, the voltage experienced at theworking electrode will lag significantly from the voltage applied. Thislag is significantly higher for a two-electrode system, as compared witha three-electrode system. In the case of two-electrode system, the valueof iR between the working and the reference electrode is significantlyhigher than that in a three-electrode system. In a three-electrodesystem, no current flows between the working electrode and the referenceelectrode, and hence the voltage drop is lower. Therefore, once thecharging current (Faradaic current) decays to a minimum (within two tothree milliseconds), the current observed is all catalytic current. In atwo-electrode system, the charging current is not diminished until thevoltage at the working electrode attains a steady state (reaches theapplied voltage). Thus, in a two-electrode system, there is a slow decayof the response profile.

The passage of the electrochemical cell can be filled with a liquidsample by any of numerous methods. Filling can be carried out by, forexample, capillary attraction, chemically-aided wicking, or vacuum.Alternatively, the liquid sample can flow through the passage. Themanner of filling the electrochemical cell depends on the application,such as single use of the sensor or continuous measurements in a flowinjection analysis.

The advantages of the invention described herein include the ability touse small volumes of liquid samples, improved current distribution, anda plurality of working electrodes.

Benefits provided by the electrochemical cell of this invention includethe capability of using low volumes of biological samples, thecapability of filling the electrochemical cell by capillary attractionor gravitational action; the capability of excluding large species ifthe dimensions of the apertures of the passages are sufficiently small;the capability of carrying out a plurality of measurements, includingmeasurement of different electroactive species. The electrochemical cellof this invention can be used in several ways, such as, for example,with a device for forming an opening in the skin by having a lancingdevice traversing the passage of the electrochemical cell when the cellis placed against the skin and allowing the electrochemical cell to befilled with a liquid sample when the liquid sample flows directly fromthe site of the opening thus formed. Alternatively, the electrochemicalcell can be used in the manner of a flow cell, with liquid sampletraversing the passage(s) under such fluid transfer techniques asconvection, diffusion, or osmosis.

The following non-limiting examples further illustrate theelectrochemical cell of this invention.

EXAMPLE 1

This example illustrates a multi-layer electrochemical cell for thedetermination of glucose. The conducting layers were formed on one majorsurface of an insulating substrate (polyvinyl chloride, approximately450 micrometers thick). A conducting layer (carbon, approximately 15 μmthick) was deposited on a PVC substrate by means of screen-printing. Thedimension of the individual cell after being trimmed was 5 mm wide and40 mm long. However, in the actual case, a plurality of cells wereprepared in one card and then cut into discrete cells having thedimensions described. The reagents, which consisted of glucose oxidase,ferrocene, and carbon (containing BES buffer, Clerol® antifoaming agent(Henkel-Nopco, Leeds, UK), and alginate binder), were screen-printed ata coating thickness of about 20 μm over a portion of the conductinglayer of carbon. A Sericard® insulating layer (approximately 20 μmthick) was screen-printed over the entire area of the conducting layer,thereby leaving a small area of the conducting layer near one of theends of the major surface of the insulating substrate exposed tofunction as an electrical contact. A layer of a mixture of silver andsilver chloride (approximately 20 μm thick) was printed over theinsulating layer to form a second conducting layer. A second Sericard®insulating layer (approximately 20 μm thick) was printed over theconducting layer of silver/silver chloride such that a portion of thesilver/silver chloride layer was allowed to remain exposed to functionas an electrical contact. The exposed portions of the two conductinglayers were used for the removable connection of the electrochemicalcell to a measuring device. The measuring device was a homemadepotentiostat capable of applying a potential to an electrochemical celland measuring the current produced. Such a device can be readily adaptedfrom a commercially available potentiostat by one of ordinary skill inthe art. A cylindrical passage having a diameter of 3 mm was formed bymeans of a mechanical punch at the area where the reagents weredeposited, such that the diameter of the passage was smaller than thearea on which the reagents were deposited. The conducting layer ofcarbon bearing the layer of reagents formed the working electrode andconducting layer containing silver/silver chloride formed thedual-purpose reference/counter electrode of the electrochemical cell.

EXAMPLE 2

This example illustrates a multi-layer electrochemical cell for thedetermination of glucose. The conducting layers were formed on bothmajor surfaces of an insulating substrate. A conducting layer comprisinga mixture of carbon, glucose oxidase, and ferrocene was applied to afirst major surface of a Melinex® insulating substrate by means ofscreen-printing so as to substantially cover the surface of the Melinex®insulating substrate, while leaving a portion on one of the ends of thefirst major surface of the Melinex® insulating substrate exposed to forman electrical contact area to allow removable connection to a measuringdevice. An insulating layer was then applied over the conducting layerby means of screen-printing in such a manner as to leave an areaadjacent to the exposed portion of the insulating substrate exposed toform an electrical contact area to allow removable connection to ameasuring device. A layer comprising a mixture of silver and silverchloride was then applied to the second major surface of the Melinex®insulating substrate by means of screen-printing in a pattern similar tothat of the conducting layer comprising carbon. The portion of the majorsurface of the insulating substrate directly opposite to the contactarea was not covered by the layer containing the mixture of silver andsilver chloride. The insulating layer was applied by means ofscreen-printing onto the layer containing silver/silver chloride in sucha manner as to allow the area adjacent to area printed area and oppositeto the area not printed on the conducting layer to remain exposed. Thisexposed area allows the electrochemical cell to make electrical contactwith the measuring device when inserted to the measuring device. Apassage was formed in the electrochemical cell assembly by punching ahole having a diameter of 4 mm through all layers by means of amechanical punch. The portion of the conducting layers thus exposed tothe passage formed the electrodes of the electrochemical cell.

The dimensions of the components in the electrochemical cell of thisexample were substantially similar to those of the electrochemical celldescribed in Example 1.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

1. An electrochemical cell comprising: an insulating substrate having aplurality of layers thereon, said layers comprising: at least twoconducting layers, wherein one of said conducting layers is a workingelectrode, said working electrode in contact with at least one reagent,wherein said at least one reagent comprises an enzyme; and at least twoinsulating layers, wherein said insulating substrate or at least one ofsaid at least two insulating layers is interposed between said at leasttwo conducting layers, wherein each major surface of each conductinglayer is in contact with a major surface of said insulting substrate ora major surface of at least one of said at least two insulating layers;and a substantially uniform passage extending from a first outer side ofthe electrochemical cell to a second outer side of the electrochemicalcell opposite the first outer side, the passage being formed througheach of said at least two conducting layers and each of said at leasttwo insulating layers to expose edges of each of said at least twoconducting layers and each of said at least two insulating layers, saidedges collectively forming a wall or walls of said passage, said exposededges of said at least two conducting layers forming said workingelectrode and a second electrode of said electrochemical cell, thepassage capable of receiving a liquid sample.
 2. The electrochemicalcell of claim 1, wherein said electrochemical cell comprises twoconducting layers and two insulating layers.
 3. The electrochemical cellof claim 2, further including a third conducting layer and a thirdinsulating layer.
 4. The electrochemical cell of claim 1, furtherincluding a second working electrode.
 5. The electrochemical cell ofclaim 4, wherein said working electrodes are capable of determining thepresence of, or the concentration of, the same analyte.
 6. Theelectrochemical cell of claim 4, wherein said working electrodes arecapable of determining the presence of, or the concentration of,different analytes.
 7. The electrochemical cell of claim 1, wherein oneof said at least two conducting layers is a counter electrode.
 8. Theelectrochemical cell of claim 1, wherein one of said at least twoconducting layers is a reference electrode.
 9. The electrochemical cellof claim 1, wherein one of said at least two conducting layers is adual-purpose reference/counter electrode.
 10. The electrochemical cellof claim 1, said at least one passage has a volume not exceeding 1microliter.
 11. The electrochemical cell of claim 1, wherein saidpassage has a regular shape.
 12. The electrochemical cell of claim 1,wherein said passage has an irregular shape.
 13. The electrochemicalcell of claim 1, wherein said at least one reagent is integral with saidworking electrode.
 14. The electrochemical cell of claim 1, wherein thethickness of each conducting layer does not exceed 100 micrometers. 15.The electrochemical cell of claim 1, wherein the thickness of eachinsulating layer does not exceed 100 micrometers.
 16. Theelectrochemical cell of claim 1, wherein said insulating substrate isinterposed between two conducting layers.
 17. The electrochemical cellof claim 1, wherein said at least one insulating layer is interposedbetween two conducting layers.
 18. The electrochemical cell of claim 1,wherein a passage is formed through said insulating substrate.